The prognosis for survival in patients with metastatic melanoma has not changed in the last 20 years and remains dismal despite advances in tumor detection and the development of melanoma-specific systemic therapies. Because of the failure of current chemotherapeutic and immunologically- based treatments to eradicate melanoma, we propose a new approach. In this proposal, we test a replication-competent oncolytic virus generated in this lab at Yale, VSVrp30. In preliminary tests VSVrp30 shows considerable promise in the potential treatment of melanoma. In vitro and in vivo experiments show that the virus selectively and rapidly infects and destroys human metastatic melanoma, with relatively little or no infection of normal human melanocytes. We seek funding to pursue experiments to determine if the virus can target and destroy melanoma cells in multiple conditions in animal and in vitro models. We will first test the oncolytic actions of the virus with a series of in vitro experiments on a large number of human melanomas and normal melanocytes available at Yale. Another set of experiments will employ human melanoma that is stably transfected with a coral reporter gene that generates a red fluorescence in the cancer cells. These human cells will be transplanted into SCID mice, both as a solid tumor, and as dispersed metastatic-like cancer cells. Virus will be given intratumorally and intravenously to test the hypothesis that the virus wll target and kill the red tumor cells with minimal infection of normal cells. Infected cells can be readily detected by the expression of a GFP reporter incorporated into the viral genome. A third set of parallel experiments will be done using the mouse melanoma line B16 in syngeneic C57Bl/6 mice with a normal immune system, allowing us to test the hypothesis that the virus can selectively detect and destroy melanoma in the presence of a normal immune system, and prolong mouse survival from melanoma; if the virus does not completely eliminate the melanoma cells, we will test the secondary hypothesis that temporarily depressing the systemic or innate immune systems with immunosuppressant drugs will enhance the oncolytic actions of the virus. A fourth set of experiments will examine the genetic mechanisms underlying the increased viral infection of melanoma cells, using an extensive series of human melanomas in which the exomes have been sequenced. These experiments will be complemented by experiments to test the hypothesis that specific induced gene mutations involving BRAF, PTEN, and CDKN2A that are common to melanoma, directly increase virus infection. A final series of experiments will test the hypothesis that the virus can cross the blood brain barrier and selectively destroy melanoma in the mouse brain, and that pre-immunization, potentially followed by transient immunosuppression, will enhance oncolysis and provide another layer of protection to the brain. If we detect collateral damage to normal brain, then we will test a new virus, 1,2-VSV, that we recently generated which is the most attenuated of any VSV we have worked with, yet still targets melanoma. Its highly attenuated nature reduces concerns relating to infection of normal brain tissue. If these experiments are successful, they will form a major advance toward clinical trials for metastatic melanoma in humans.